Persistent colonization of the human stomach with the Gram-negative bacterium Helicobacter pylori is associated with an increased risk for development of gastric adenocarcinoma and peptic ulcer disease. The long-term objective of this project is to understand the molecular mechanisms by which H. pylori contributes to these diseases, and to develop effective means for the prevention and treatment of these diseases. An important virulence factor produced by H. pylori is a secreted toxin known as VacA. Studies in a mouse model indicate that VacA enhances the capacity of H. pylonto colonize the stomach and that it can cause gastric epithelial damage. Analyses of vacA allelic variation in H. pylori isolates from humans suggest that VacA plays a role in the pathogenesis of gastric cancer and peptic ulcer disease. In vitro studies indicate that VacA is a multifunctional toxin. VacA causes multiple alterations in human gastric epithelial cells, including swelling of endosomal compartments (cell vacuolation) and changes in mitochondria! function. In addition to its effects on epithelial cells, VacA inhibits activation and proliferation of T lymphocytes. A current model proposes that VacA intoxicates gastric epithelial cells through a multi- step process, which includes binding of the toxin to the plasma membrane, oligomerization, membrane insertion, membrane channel formation, internalization, intracellular trafficking, and localization in specific intracellular sites. The central hypotheses of the current proposal are that VacA has unique structural features, and that actions required for individual steps in the intoxication process can be mapped to different regions of the VacA protein. In Aim #1 of this proposal, we will analyze the crystal structure of VacA. In Aim #2 we will map regions of VacA that have specific functional activities. In Aim #3, we will analyze the role of VacA in H. pylori colonization of the stomach. These studies should lead to important advances in our understanding of the biology of H. pylori- host interactions, and should ultimately lead to advances in the treatment and prevention of H. py/or/-associated human diseases. Relevance to public health: The presence of a bacterium known as Helicobacter pylori in the human stomach contributes to the development of cancer of the stomach and peptic ulcer disease. This research seeks to understand how a bacterial infection can lead to these diseases, and seeks to develop new approaches for the prevention and therapy of stomach cancer and peptic ulcer disease.